1. Field of the Invention
The present invention discloses a method and composition for the treatment of bacterial infections by the use of a lytic enzymes and holin enzymes blended with an appropriate carrier suitable for the treatment of the infection.
2. Description of the Prior Art
In the past, antibiotics have been used to treat various infections. The work of Selman Waksman in the introduction and production of Streptomycetes, Dr. Fleming""s discovery of penicillin, are well known as well as the work of numerous others in the field of antibiotics. Over the years, there have been additions and chemical modifications to the xe2x80x9cbasicxe2x80x9d antibiotics in attempts to make them more powerful, or to treat people allergic to these antibiotics.
Others have found new uses for these antibiotics. U.S. Pat. No. 5,260,292 (Robinson et al.) discloses the topical treatment of acne With aminopenicillins. The method and composition for topically treating acne and acneiform dermal disorders includes applying an amount of an antibiotic selected from the group consisting of amnpicillin, amoxicillin, other aminopenicillins, and cephalosporins, and derivatives and analogs thereof, effective to treat the acne and acneiform dermal disorders. U.S. Pat. No. 5,409,917 (Robinson et al.) discloses the topical treatment of acne with cephalosporins.
However, as more antibiotics have been prescribed or used at an ever increasing rate for a variety of illnesses, increasing numbers of bacteria have developed a resistance to antibiotics. Larger doses of stronger antibiotics are now being used to treat ever more resistant strains of bacteria. Multiple antibiotic resistant bacteria have consequently developed. The use of more antibiotics and the number of bacteria showing resistance has led to increasing the amount of time that the antibiotics need to be used. Broad, non-specific antibiotics, some of which have detrimental effects on the patient, are now being used more frequently. Also, antibiotics do not easily penetrate mucus linings. Additionally, the number of people allergic to antibiotics appears to be increasing.
Consequently, other efforts have been sought to first identify and then kill bacteria.
Attempts have been made to treat bacterial diseases with by the use of bacteriophages. U.S. Pat. No. 5,688,501 (Merril, et al.) discloses a method for treating an infectious disease caused by bacteria in an animal with lytic or non-lytic bacteriophages that are specific for particular bacteria.
U.S. Pat. No. 4,957,686 (Norris) discloses a procedure of improved dental hygiene which comprises introducing into the mouth bacteriophages parasitic to bacteria which possess the property of readily adhering to the salivary pellicle.
It is to be noted that the direct introduction of bacteriophages into an animal to prevent or fight diseases has certain drawbacks. Specifically, the bacteria must be in the right growth phase for the phage to attach. Both the bacteria and the phage have to be in the correct and synchronized growth cycles. Additionally, there must be the right number of phages to attach to the bacteria; if there are too many or too few phages, there will either be lysis from without or no production of the lysing enzyme, respectively. The phage must also be active where damaged phase are inactive. The phages are also inhibited by many things including bacterial debris from the organism it is going to attack. Further complicating the direct use of bacteriophage to treat bacterial infections is the possibility of immunological reactions, rendering the phage non-functional.
Consequently, others have explored the use of other safer and more effective means to treat and prevent bacterial infections.
U.S. Pat. No. (application Ser. No. 08/962,523) (Fischetti, et. al.) and U.S. Pat. No. (application Ser. No. 09/257,026) (Fischetti et al.) disclose the use of an oral delivery mode, such as a candy, chewing gum, lozenge, troche, tablet, a powder, an aerosol, a liquid or a liquid spray, containing a lysin enzyme produced by group C streptococcal bacteria infected with a C1 bacteriophage for the prophylactic and therapeutic treatment of Streptococcal A throat infections, commonly known as strep throat.
U.S. Pat. No. 09/395,636 (Fischetti et al.) discloses a method and composition for the prophylactic or therapeutic treatment of bacterial infections, comprising administering an effective amount of at least one lytic enzyme produced by a bacteria infected with a bacteriophage specific for the bacteria to the site of the infection. The lytic enzyme preferably comprises a carrier suitable for delivering the lytic enzyme to the site of the infection. This method and treatment may be used for treating upper respiratory infections, topical infections, vaginal infections, eye infectionsear infections, for parenteral treatment, and for most other bacterial infections.
The method for obtaining and purifying the lytic enzyme produced by a bacteria infected with the bacteriophage is known in the art. While many lytic enzymes are phage encoded, some recent evidence suggests that phage enzymes may actually be a bacterial enzyme that is used to construct the cell wall and the phage. While replicating in the bacterium, a phage gene product may cause the upregulation or derepression of bacterial enzyme for the purpose of releasing the bacteriophage. These bacterial enzymes may be tightly regulated by the bacterial cell and are used by the bacteria for the construction and assembly of the cell wall.
The use of these lytic enzymes for the prophylactic and therapeutic treatment of bacterial diseases, however, has not been explored, except by the inventors of the present invention. Consequently, the present invention discloses the extraction and use of a variety of bacterial phage associated lytic enzymes, holin enzymes, chimeric enzymes, and shuffled enzymes for the treatment of a wide variety of illnesses caused by bacterial infections.
The use of phage associated lytic enzymes produced by the infection of a bacteria with a bacteria specific phage has numerous advantages for the treatment of diseases. As the phage are targeted for specific bacteria, the lytic enzymes do not interfere with normal flora. The actions of the lytic enzymes are fast and do not depend on bacterial growth.
Lytic enzymes can be directed to the mucosal lining, where, in residence, they will be able to kill colonizing disease bacteria.
Shuffled enzymes are enzymes in which the gene for more than one related lytic enzymes have been randomly cleaved and reassembled into a more active or specific enzyme.
In a preferred embodiment of the invention, shuffled enzymes are used to treat bacterial infections, thereby increasing the speed and efficiency with which the bacteria are killed.
Chimeric enzymes are enzymes which are a combination of two or more enzymes having two or more active sites such that the chimeric enzyme can act independently on the same or different molecules. This will allow for potentially treating two or more different bacterial infections at the same time. Chimeric enzymes may also be used to treat one bacterial infection by cleaving the cell wall in more than one location.
A number of chimeric lytic enzymes have been produced and studied. Gene E-L, a chimeric lysis constructed from bacteriophages phi X174 and MS2 lysis proteins E and L, respectively, was subjected to internal deletions to create a series of new E-L clones with altered lysis or killing properties. The lytic activities oft he parental genes E, L, E-L, and the internal truncated forms of E-L were investigated in this study to characterize the different lysis mechanism, based on differences in the architecture of the different membranes spanning domains. Electron microscopy and release of marker enzymes for the cytoplasmic and periplasmic spaces revealed that two different lysis mechanisms can be distinguished depending on penetrating of the proteins of either the inner membrane or the inner and outer membranes of the E. coli. FEMS Microbiol. Lett. 1998 Jul. 1, 164(1); 159-67.
In another experiment an active chimeric cell wall lytic enzyme (TSL) has been constructed by fusing the region coding for the N-terminal half of the lactococcal phage Tuc2009 lysin and the region coding for the C-terminal domain oft he major pneumococcal autolysin. The chimeric enzyme exhibited a glycosidase activity capable of hydrolysing choline-containing pneumoccal cell walls.
A preferred embodiment of this invention discloses the use of chimeric lytic enzymes to treat two infectious bacteria at the same time, or to cleave the cell wall of a bacteria in two different locations.
Holin enzymes produce holes in the cell membrane. More specifically, holins form lethal membrane lesions that terminates respiration. Like the lytic enzymes, the holin enzymes are coded for and carried by a phage. In fact, it is quite common for the genetic code for the holin enzyme to be found next to or even within the code for the lytic enzyme in the phage. Most holin sequences are short, and overall, hydrophobic in nature, with a highly hydrophilic carboxy-terminal domain. In many cases, the putative holin is encoded on a different reading frame within the enzymatically active domain of the phage. In other cases, the holin is encoded on the DNA next or close to the DNA coding for the cell wall lytic enzyme. The holin is frequently synthesized during the late stage of phage infection and found in the cytoplasmic membrane where it causes membrane lesions.
Holins can be grouped into two general classes based on primary structure analysis. Class I holins are usually 95 residues or longer and may have three potential transmembrane domains. Class II holins are usually smaller, at approximately 65-95 residues, and the distribution of charged and hydrophobic residues indicatoing two TM domains (Young, et al. Trends in Microbiology v. 8, No. 4, March 2000). At least for the phages of gram-positive hosts, however, the dual-component lysis system may not be universal. Although the presence of holins has been shown or suggested for several phages, no genes have yet been found encoding putative holins for all oft he phages. Holins have been shown to be present or suggested for among others, lactococcal bacteriophage Tuc2009, lactococcal xcfx86LC3, pneumococcal bacteriophage EJ-1, Lactobacillus gasseri bacteriophage xcfx86anh, Staphylococcus aureus bacteriophage Twort, Listeria monocytogenes bacteriophages, pneumococcal phage Cp-1, Bacillus subtillis phage "PHgr"29, Lictobacillus delbrueckki bacteriophage LL-H lysin, and bacteriophage xcfx8611 of Staphyloccous aureus. (Loessner, et al., Journal of Bacteriology, August 1999, p. 4452-4460).
In another embodiment of the invention, holin enzymes are used in conjunction with the lytic enzymes to accelerate the speed and efficientcy at which the bacteria are killed. Holin enzymes may also be in the form of chimeric and/or shuffled enzymes. Holin enzymes may also be used alone in the treatment of bacterial infections
It is an object of the invention to use phage associated lytic enzymes in combination with chimeric or shuffled lytic enzymes to prophylactically and therapeutically treat bacterial diseases.
In another embodiment of the invention, chimeric lytic enzymes are used to prophylactically and therapeutically treat bacterial diseases.
In yet another embodiment of the invention, shuffled lytic enzymes are used to prophylactically and therapeutically treat bacterial infections.
In yet another embodiment oft he invention, holin enzymes are used in conjunction with phage associated lytic enzymes to prophylactically and therapeutically treat bacterial infections.
In another embodiment oft he invention, holin enzymes alone are used to prophylactically and therapeutically treat bacterial infections.
In another embodiment of the invention, the holin enzymes are shuffled holin enzymes or chimeric holin enzymes, either in combination with or independent of the lytic enzymes.
The invention (which incorporates U.S. Pat. No. 5,604,109 in its entirety by reference) uses a lytic enzyme produced by the bacterial organism after being infected with a particular bacteriophage as either a prophylactic treatment for preventing those who have been exposed to others who have the symptoms of an infection from getting sick, or as a therapeutic treatment for those who have already become ill from the infection. The present invention is based upon the discovery that phage lytic enzymes specific for bacteria infected with a specific phage can effectively and efficiently break down the cell wall of the bacterium in question. At the same time, the semipurified enzyme is lacking in proteolytic enzymatic activity and therefore non-destructive to mammalian proteins and tissues when present during the digestion of the bacterial cell wall. As discussed above, the lytic enzymes may be chimeric, shuffled or xe2x80x9cnatural,xe2x80x9d and may be in combination with at least one holin enzyme, which may also be chimeric, shuffled, or xe2x80x9cnatural.xe2x80x9d
In one embodiment of the invention, the prophylactic and therapeutic treatment of a variety of illnesses caused by Streptococcal pyogehes, Streptococcal pneumoniae, Streptococcus fasciae, and Hemophilus influenza are disclosed. In another embodiment of the invention, gram negative bacterial infections caused by Listeria, Salmonella, E. coli, and Campylobacter, are treated by the use of lytic enzymes. These and other bacteria, which can infect the digestive system, can be treated by incorporating the lytic enzymes in suppository enemas, in syrups, or in other carriers to get directly to the site of the infection(s).
In another embodiment of the invention, lytic enzymes are incorporated into bandages to prevent or treat infections of burns and wounds. In yet another embodiment of the invention, the lytic enzymes of phage associated with Staphylococcus or Pseudomonas are incorporated into bandages to prevent or treat infections of burns and wounds.
Vaginal infections caused by Group B Streptococcus can cause premature birth and subsequent complications resulting in neonatal sepsis. Lysin incorporated into tampons specific for group B strep would prevent infection of the neonate during birth without disturbing normal vaginal flora so that women would not be overcomeiby yeast infection as a result of antibiotic therapy.
In another embodiment of the invention, eye drops containing lytic enzymes of Hemophilus, Pseudomonas, and/or Staphylococcus can be used to directly treat eye infections. Treatment with lytic enzymes are faster and more expedient,than with antibiotics.
In yet another embodiment of the invention the phage associated lytic enzyme is put into a carrier which is placed in an inhaler to treat or prevent the spread of diseases localized in the mucus lining of the oral cavity and lungs. Specific lytic enzymes for tuberculosis have been isolated and can be used.
In another embodiment of the invention the lytic enzyme is administered in the form of a candy, chewing gum, lozenge, troche, tablet, a powder, an aerosol, a liquid, a liquid spray, or toothpaste for the prevention or treatment of bacterial infections associated with upper respiratory tract illnesses.
In another embodiment of the invention, species specific lytic enzymes can be used in the treatment of bacterial infections associated with topical or dermatological infections, administered in the form of a topical ointment or cream. In another embodiment of the invention, the lytic enzyme would be administered in an aqueous form. In yet another embodiment oft he invention, lysostaphin, the enzyme which lyses Staphylococcus aureus, can be included in the therapeutic agent. In a further embodiment of the invention, conventional antibiotics may be included in the therapeutic agent with the lytic enzyme, and with or without the presence of lysostaphin. More than one lytic enzyme may also be included in the prophylactic or therapeutic agent.